FR3032203A1 - METHOD FOR INDUCING TARGETED MEIOTIC RECOMBINATIONS - Google Patents

Abstract

The present invention relates to a fusion protein comprising a Cas9 domain and a Spo11 domain, as well as the use of this protein to induce targeted meiotic recombination in a eukaryotic cell.

Description

The present invention relates to the field of eukaryotes, more particularly to the field of microbiology. In particular, it relates to a method for improving or modifying a yeast strain by inducing targeted meiotic recombinations. BACKGROUND OF THE INVENTION Yeasts find their application in extremely varied industrial fields. Due to the safety of a large number of species, yeasts are particularly used in the food industry as a fermentation agent in bakery, brewery, winemaking or distillery, or in the form of extracts as nutritional elements or agents. of palatability. They can also find their use in the industrial production of bioethanol or molecules of interest such as vitamins, antibiotics, vaccines, enzymes or steroid hormones, or in the processes of degradation of cellulosic materials. The diversity of industrial applications of yeasts implies that there is a constant demand for yeast strains with improved characteristics or at least adapted to new use or new culture conditions. In order to obtain a strain having a particular characteristic, one skilled in the art can use sexual reproduction by crossing two parental strains having interesting characteristics and by selecting a hybrid strain providing the desired combination of parental characteristics. This method is however random and the selection step can lead to significant delays. Alternatively, it can also genetically modify the strain by a recombinant DNA technique. This modification may nevertheless constitute a brake on its operation, whether for regulatory, health or environmental reasons. A third alternative is to cause reassortment of paternal and maternal alleles in the genome during meiotic recombination. Meiotic recombination is a DNA exchange between homologous chromosomes during meiosis. It is initiated by the formation of double-strand breaks in one or the other of the homologous chromatids, followed by the repair of these breaks, using as a matrix a chromatid of the homologous chromosome. Meiotic recombinations, however, have the disadvantage of being random and non-uniform. Indeed, the double-stranded cleavage sites at the origin of these recombinations are not distributed homogeneously in the genome. There are thus distinguished chromosomal regions 'hot' where the frequency of recombination is high, and chromosome regions called 'cold' where the recombination frequency can be up to 100 times lower.

Spo 11 is the protein that catalyzes double-strand breaks during meiosis. It acts as a dimer in cooperation with many partners. At present, the factors determining the choice of double-strand cleavage sites by Spoll and its partners remain poorly understood. Controlling the formation of double-strand breaks and, in fact, meiotic recombinations, is crucial for the development of genetic engineering techniques. It has recently been shown that it is possible to modify the double-strand breakage formation sites by fusing Spol 1 with the Ga14 transcription activator DNA binding domain (Pecina et al, 2002 Cell, 111). , 173-184,). The Ga14-Spoll fusion protein makes it possible to introduce double-strand breaks into so-called 'cold' chromosomal regions at the sites of Ga14 binding to DNA. However, in the latter approach, the introduction of double-strand breaks is conditioned by the presence of Ga14 binding sites, it is therefore impossible to induce meiotic recombination phenomena targeted independently of specific binding sites. SUMMARY OF THE INVENTION The object of the present invention is to provide a method for inducing targeted meiotic recombination in eukaryotic cells, preferably yeast cells, in any region of the genome, regardless of any site. known binding, and in particular in so-called 'cold' chromosomal regions. Thus, according to a first aspect, the present invention relates to a method for inducing targeted meiotic recombinations in a eukaryotic cell comprising - introducing into said cell: a) a fusion protein comprising a Cas9 domain and a Spo domain 11 or a nucleic acid encoding said fusion protein; and b) one or more guide RNAs or one or more nucleic acids encoding said guide RNAs, said guide RNAs comprising a Cas9 domain binding RNA structure of the fusion protein and a sequence complementary to the targeted chromosomal region; and - inducing the entry into prophase I of meiosis of said cell. The fusion protein may further comprise a nuclear localization signal sequence.

Preferably, the Cas9 domain of the fusion protein is a Cas9 protein deficient for nuclease activity. The nucleic acid encoding said fusion protein may be placed under the control of a constitutive, inducible or meiosis specific promoter. One or more additional guide RNAs targeting one or more other chromosomal regions, or nucleic acids encoding said additional guide RNAs, may be introduced into the eukaryotic cell. Preferably, the eukaryotic cell is a yeast. The entry into prophase I of the yeast can then be induced by its transfer from a rich medium to a sporulation medium. The present invention also relates, in a second aspect, to a fusion protein as defined in the above process. According to a third aspect, the present invention further relates to a nucleic acid encoding the fusion protein defined above. The present invention also relates, according to a fourth aspect, to an expression cassette or a vector comprising a nucleic acid as defined above. Preferably, the vector is a plasmid comprising, an origin of bacterial replication, an expression cassette comprising a nucleic acid as defined above, one or more selection markers, and / or one or more sequences allowing insertion. target of the vector, expression cassette or nucleic acid in the genome of the host cell. In particular, the plasmid comprises an origin of bacterial replication, preferably the Co1E1 origin, an expression cassette comprising a nucleic acid as defined above under the control of a promoter, preferably the ADH1 promoter, a terminator , preferably the ADH1 terminator, one or more selection markers, preferably resistance markers such as the kanamycin or ampicillin resistance gene, one or more sequences allowing the targeted insertion of the vector, the cassette expression or nucleic acid in the genome of the host cell, preferably at the TRP 1 locus of the genome of a yeast. Preferably, the plasmid comprises, or consists of, a nucleotide sequence selected from SEQ ID NO: 1 and SEQ ID NO: 2. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1. Schematic diagram showing plasmids P1 (SEQ ID NO: 1) and P2 (SEQ ID NO: 2) of the SpCas9-Spo1 1 or SpCas9 * -Spo1 fusion 1. P1 and P2 respectively code for the expression of NLS-SpCas9-Spo1 1 and NLS-SpCas9 * -Spo 1 1 in yeast. The black blocks represent the constitutive promoter ADH1 (pADH1) and the terminator ADH1 (tADH1). The arrow indicates the direction of transcription under the control of the ADH1 promoter. Figure 2. Diagram showing gRNA expression plasmids in yeast cells. (A) Schematic of the plasmid containing a single gRNA expression cassette to target a single region of the yeast genome. RE indicates the restriction site where the sequence determining the gRNA specificity (SDS) is inserted by the Gibson method. The promoter and terminator (Term) are dependent on RNA polymerase III. The arrow on the promoter indicates the direction of transcription of the sequence. The expression cassette of a gRNA contains a gRNA flanked by a promoter and a terminator. (B) Diagram of the plasmid containing several gRNA expression cassettes to target multiple regions of the yeast genome (multiplex targeting). The different gRNA cassettes are distinguished by their sequence determining their specificity (SDS). They were introduced successively into the multiple cloning site (MCS) by conventional cloning / ligation techniques. Figure 3. Schematic representing the plasmid P1 (SEQ ID NO: 1). Figure 4. Diagram showing plasmid P2 (SEQ ID NO: 2). DETAILED DESCRIPTION OF THE INVENTION CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats) System is a bacterial defense system against foreign DNA. This system is essentially based on the combination of a Cas9 protein and a "guide" RNA (gRNA) responsible for the specificity of the cleavage site. It allows the realization of double-stranded DNA breaks (CDB) at sites targeted by the CRISPR / Cas9 system. This system has already been used to carry out targeted genome engineering in eukaryotic cells (see, for example, patent application EP2764103), especially human cells (Cong L et al., 2013, Science 339 (6121): 819-823, Mali P et al., 2013, Science, 339 (6121): 823-826, Cho SW et al., 2013, Nature Biotechnology 31 (3): 230-232), Rat (Li D, and al., 2013, Nature Biotechnology, 31 (8): 681-683; WO 2014/089290), mouse (Wang H et al., 2013, Cell, 153 (4): 910-918), rabbit (Yang D et al., 2014, Journal of Molecular Cell Biology, 6 (1): 97-99), Frog (Nakayama T et al., 2013, Genesis, 51 (12): 835-843), Fish (Hwang WY et al., 2013, Nature Biotechnology, 31 (3): 227-229), plants (Shan Q et al., 2013, Nature Biotechnology, 31 (8): 686-688, Jiang W et al., 2013 , Nucleic Acids Research, 41 (20): e188.), Drosophila (Yu Z et al, 2013, Genetics, 195 (1): 289-291), nematode (Friedland AE et al., 2013, Nature Methods, 10 (8): 741-743), yeast (DiCarlo J, et al. 2013, Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. Nucleic Acids Research 41 (7): 4336-4343), but also bacterial cells (Jiang W et al., 2013, Nature Biotechnology, 31 (3): 233-239). In contrast, this system has never been used to target meiotic recombination sites in any organism. The inventors have demonstrated that it is possible to modify the CRISPR-Cas9 system in order to induce targeted meiotic recombinations in a eukaryotic cell, and in particular in a yeast. They have indeed shown that the combined expression of a Spoll-Cas9 fusion protein and one or more guide RNAs makes it possible to target the action of the Spo 11 transesterase which is responsible for double-strand breaks during meiosis. The repair of these breaks by using as a matrix a chromatide of the homologous chromosome induces the desired recombination phenomena. Thus, the present invention relates to a method for inducing targeted meiotic recombination in a eukaryotic cell comprising - introducing into said cell: a) a fusion protein comprising a Cas9 domain and a Spo domain 11, or an acid nucleic acid encoding said fusion protein; and b) one or more guide RNAs or one or more nucleic acids encoding said guide RNAs, said guide RNAs comprising a Cas9 domain binding RNA structure of the fusion protein and a sequence complementary to the targeted chromosomal region; and - inducing the entry into prophase I of meiosis of said cell. As used herein, the term "eukaryotic cell" refers to a yeast, plant, fungus or animal cell, in particular a mammalian cell such as a mouse or rat cell, or a insect cell. The eukaryotic cell is preferably non-human and / or non-embryonic. According to a particular embodiment, the eukaryotic cell is a yeast cell, in particular a yeast of industrial interest. Examples of yeasts of interest include, but are not limited to, yeasts of the genus Saccharomyces sensu stricto, Schizosaccharomyces, Yarrowia, Hansenula, Kluyveromyces, Pichia or Candida, as well as hybrids obtained from a strain belonging to one of these genres. Preferably, the yeast of interest belongs to the genus Saccharomyces. It may especially belong to a species selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces bayanus, Saccharomyces castelli, Saccharomyces eubayanus, Saccharomyces kluyveri, Saccharomyces kudriavzevii, Saccharomyces mikatae, Saccharomyces uvarum, Saccharomyces paradoxus and Saccharomyces pastorianus (also called Saccharomyces carlsbergensis), or is a hybrid obtained from a strain belonging to one of these species such as, for example, a S. cerevisiae / S. paradoxus hybrid or a S. cerevisiae / S hybrid. uvarum. According to another particular embodiment, the eukaryotic cell is a mushroom cell, in particular a mushroom cell of industrial interest. Examples of fungi include, but are not limited to, filamentous fungi cells. Filamentous fungi include fungi belonging to the Eumycota and Oomycota subdivisions. The filamentous fungal cells may be selected from the group consisting of Trichoderma, Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora cells. , Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium or Trametes. As used herein, the term "fusion protein" refers to a chimeric protein comprising at least two domains derived from the combination of different proteins or protein fragments. The nucleic acid encoding this protein is obtained by recombining the regions encoding the proteins or protein fragments so that they are in phase and transcribed on the same mRNA. The different domains of the fusion protein can be directly adjacent or separated by linker sequences that introduce some structural flexibility into the construct. The fusion protein used in the present invention comprises a Cas9 domain and a Spoll domain. The Cas9 domain is the domain of the fusion protein that is capable of interacting with the guide RNAs and targeting the nuclease activity of the Spo 11 domain to a given chromosomal region. The Cas9 domain may consist of a wild-type or modified Cas9 protein (also called Csn1 or Csx12), or a fragment of this protein capable of interacting with the guide RNAs. In particular, the Cas9 protein may be modified to modulate its enzymatic activity. Thus, the nuclease activity of the Cas9 protein can be modified or inactivated. The Cas9 protein may also be truncated to remove non-essential protein domains from the fusion protein functions, particularly those domains of the Cas9 protein that are not required for interaction with the guide RNAs. The Cas9 protein or fragment thereof as used in the present invention can be obtained from any known Cas9 protein (Makarova et al., 2008, Nat Rev Microbiol., 9, pp. 466-477). ). Examples of Cas9 proteins useful in the present invention include, but are not limited to, Cas9 proteins of Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., Nocardiopsis ssonvillei, Streptomyces pristinaespiralis, Streptomyces viridochromogenes, Streptomyces viridochromogenes, Streptosporangium roseum, Streptosporangium roseum, Alicyclobacillus acidocaldarius, Bacillus pseudomycoides, Bacillus selenitireducens, Exiguobacterium sibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius, Microscilla marina, Burkholderiales bacterium, Polaromonas naphthalenivorans, Polaromonas sp., Crocosphaera watsonii, Cyanothec sp., Microcystis aeruginosa, Synechococcus sp., Acetohalobium arabaticum, Ammonifex degensii , Caldicelulosiruptor becscii, Candidatus Desulforudis, Clostridium botulinum, Clostridium difficile, Finegoldia magna, Natranaerobius thermophilus, Pelotomaculum thermopropionicum, Acidithiobacillus caldus, Acidithiobacillus ferrooxidans, Allochromatium vinosum, Marinobacter sp., Nitrosococcus halophilus, Nitrosococcus watsoni, Pseudoalteromonas haloplanktis, Ktedonobacter racemifer, Methanohalobium evestigatum, Anabaena variabilis, Nodularia spumigena, Nostoc sp., Arthrospira maxima, Arthrospira platensis, Arthrospira sp., Lyngbya sp., Microcoleus chthonoplasts, Oscillatoria sp., Petrotoga mobilis, Thermosipho africanus, or Acaryochloris marina. Other Cas9 proteins usable in the present invention are also described in the article by Makarova et al. (Makarova et al., 2008, Nat Rev. Microbiol., 9, pp. 466-477). Preferably, the Cas9 domain comprises, or consists of, the Cas9 protein of Streptococcus pyogenes (NCBI accession number: WP 010922251.1, SEQ ID NO: 8) or a fragment thereof capable of interacting with the guide RNAs. According to a particular embodiment, the Cas9 domain consists of an entire Cas9 protein, preferably the Cas9 protein of Streptococcus pyogenes. In general, the Cas9 proteins comprise two nuclease domains: a domain-related domain RuvC and a domain-related domain HNH. These two domains cooperate to create double-strand breaks in DNA (Jinek et al., Science, 337: 816-821). Each of these nuclease domains can be inactivated by deletion, insertion or substitution using techniques well known to those skilled in the art such as site-directed mutagenesis, PCR mutagenesis or total gene synthesis. Thus, the RuvC domain can be inactivated for example by the D 10A substitution and the HNH domain can be inactivated, for example by the H840A substitution (Jinek et al., Science, 337: 816-821), the positions indicated being those of the SEQ ID NO: 8. In the peptide sequences described in this document, the amino acids are represented by their one-letter code according to the following nomenclature: C: cysteine; D: aspartic acid; E: glutamic acid; F: phenylalanine; G: glycine; H: histidine; I: isoleucine; K: lysine; L: leucine; M: methionine; N: asparagine; P: proline; Q: glutamine; R: arginine; S: serine; T: threonine; V: valine; W: tryptophan and Y: tyrosine. According to one embodiment, the Cas9 domain is deficient for at least one nuclease activity. This domain can be obtained by inactivating at least one of the nuclease domains of the Cas9 protein as described above. According to a particular embodiment, the Cas9 domain comprises, or consists of, a Cas9 protein or a Cas9 protein fragment, lacking nuclease activity (also called Cas9 *). This catalytically inactive form can be obtained by inactivating the two nuclease domains of the Cas9 protein as mentioned above, for example by introducing the two point mutations substituting aspartate 10 and histidine 840 with alanines. According to a preferred embodiment, the Cas9 domain comprises, or consists of, a Cas9 protein, preferably the Cas9 protein of Streptococcus pyogenes, devoid of nuclease activity. According to a particular embodiment, the Cas9 domain comprises, or consists of, the sequence presented in SEQ ID NO: 8 in which the aspartate at position 10 and the histidine at position 840 have been substituted with alanines. Spo 11 is a protein related to the catalytic A subunit of a type II topoisomerase present in archaebacteria (Bergerat et al., Nature, 386, pp 414-7). It catalyzes the double-strand breaks of DNA initiating meiotic recombination. It is a highly conserved protein for which there are homologues in all eukaryotes. Spoll is active as a dimer consisting of two subunits, each of which cleaves a strand of DNA. Although essential, Spol 1 does not act alone to generate double-strand breaks during meiosis. In S. cerevisiae yeast, for example, it cooperates with proteins, Rec102, Rec103 / 5k18, Rec104, Rec114, Merl, Mer2 / Rec107, Mei4, Mre2 / Nam8, Mrell, Rad50, Xrs2 / Nbs1, Hopi, Redl, Mekl, Sen and Sppl as well as other partners described in the articles by Keeney et al. (2001, Curr Top, Dey Biol, 52, pp. 1-53), Smith et al. (Curr Opin Genet Dey, 1998, 8, pp. 200-211) and Acquaviva et al. (2013 Science, 339, pp. 215-8). It has recently been demonstrated, however, that spoll targeting at a given site is sufficient to trigger the meiotic recombination process (Pecina et al, 2002 Cell, 111, 173-184). The Spo 11 domain of the Cas9-Spo 11 fusion protein is generally the domain responsible for double-strand breaks. This domain may consist of a Spol 1 protein or a fragment thereof capable of inducing double-strand breaks of the DNA.

The Spoll protein or fragment thereof as used in the present invention can be obtained from any known Spoll protein such as Saccharomyces cerevisiae Spoll protein (Gene ID: 856364, NCBI accession number: NP 011841 (SEQ ID NO: 9) Esposito and Esposito, Genetics, 1969, 61, pp. 79-89), AtSpo 1 1-1 protein and AtSpo 1-2-2 of Arabidopsis thaliana (Grelon M. et al, 2001). Embo J., 20, pp. 589-600), mSpo murine protein 11 (Baudat F et al, Molecular Cell, 2000, 6, pp. 989-998), C. elegans spool protein, or protein Drosophila Spoll meiW68 (McKim et al, 1998, Genes Dey, 12 (18), pp. 2932-42). Of course, these examples are not limiting and any known Spoll protein can be used in the process according to the invention. According to a preferred embodiment, the Spo domain 11 comprises, or consists of, a Spoll protein, preferably a Spoll protein of Saccharomyces cerevisiae, such as, for example, the protein of sequence SEQ ID NO: 9. According to a particular embodiment the Spo domain 11 is deficient for nuclease activity. In particular, the Spo domain 11 may comprise, or consist of, the mutant protein Spo 11-Y135F, a mutant protein incapable of inducing double-strand breaks of the DNA (Neale MJ, 2002, Molecular Cell, 9, 835-846). The position indicated is that of SEQ ID NO: 9. The ability of the fusion protein according to the invention to induce double-strand breaks of the DNA can come from the Cas9 domain or the Spol domain 1. Thus, the protein method of fusion comprises at least one domain, Cas9 or Spoll, having nuclease activity, preferably the Spoll domain. The fusion protein according to the invention comprises a Spoll domain and a Cas9 domain as defined above. According to one embodiment, the Spoll domain is on the N-terminal side and the Cas9 domain is on the C-terminal side of the fusion protein. In another embodiment, the Spoll domain is on the C-terminal side and the Cas9 domain is on the N-terminal side of the fusion protein. The fusion protein may also include a nuclear localization signal (SLN) sequence. SLN sequences are well known to those skilled in the art and generally comprise a short sequence of basic amino acids. By way of example, the SLN sequence may comprise the sequence PKKKRKV (SEQ ID NO: 3). The SLN sequence may be present at the N-terminus, C-terminus or in an internal region of the fusion protein, preferably at the N-terminus of the fusion protein.

The fusion protein may also include a further domain of penetration into the cell, i.e., a domain facilitating entry of the fusion protein into the cell. This type of domain is well known to those skilled in the art and may comprise, for example, a peptide penetration sequence derived from the HIV-1 TAT protein such as GRKKRRQRRRPPQPKKKRKV (SEQ ID NO: 4), derived from the TLM sequence of the virus. human hepatitis B such as PLSSIFSRIGDPPKKKRKV (SEQ ID NO: 5), or a polyarginine peptide sequence. This domain of penetration into the cell may be present at the N-terminus, C-terminus or be within the fusion protein, preferably at the N-terminus. The fusion protein may further comprise one or more linker sequences between the Cas9 and Spoll domains, and optionally between these domains and the other domains of the protein such as the nuclear localization signal sequence or the domain of penetration into the cell. The length of these binding sequences is easily adjustable by those skilled in the art. In general, these sequences comprise between 10 and 20 amino acids, preferably about 15 amino acids and more preferably 12 amino acids. The linking sequences between the different domains may be of identical or different lengths. According to a particular embodiment, the fusion protein comprises, or consists of, successively, from the N-terminus to the C-terminus: a nuclear localization signal, a first linker sequence (linkerl) , a Cas9 domain, a second linker2 (linker2) and a Spoll domain. According to another particular embodiment, the fusion protein comprises, or consists of, successively, from the N-terminus to the C-terminus: a nuclear localization signal, a first linker sequence (linker ), a Spo domain 11, a second linker2 (linker2) and a Cas9 domain. The fusion protein may further comprise a tag (or tag) which is a defined sequence of amino acids. This tag can in particular be used to detect the expression of the fusion protein, to identify the proteins interacting with the fusion protein or to characterize the sites of attachment of the fusion protein in the genome. The detection of the label attached to the fusion protein may be carried out with an antibody specific for said label or any other technique well known to those skilled in the art. Identification of the proteins interacting with the fusion protein can be achieved, for example, by co-immunoprecipitation techniques. Characterization of the binding sites of the fusion protein in the genome can be performed, for example, by immunoprecipitation techniques, chromatin immunoprecipitation coupled to real-time quantitative PCR (ChIP-qPCR), immunoprecipitation of chromatin coupled to sequencing techniques (ChIP-Seq), mapping using oligonucleotides (oligo mapping) or any other technique well known to those skilled in the art. This tag may be present at the N-terminus of the fusion protein, at the C-terminus of the fusion protein, or in the non-terminal position in the fusion protein. Preferably, the tag is present at the C-terminus of the fusion protein. The fusion protein can comprise one or more labels, which are identical or different. The labels as used in the present invention may be selected from the many labels well known to those skilled in the art. In particular, the labels used in the present invention may be peptide labels and / or protein labels. Preferably, the tags used in the present invention are peptide tags. Examples of peptide labels for use in the present invention include, but are not limited to, repetitive labels of at least six Histidines (His), particularly six or eight histidine labels, as well as Flag labels, polyglutamates, haemagglutinin (HA), calmodulin, Strep, E-tag, myc, V5, Xpress, VSV, S-tag, Avi, SBP, Softag 1, Softag 2, Softag 3, isopetag, SpyTag, tetracysteines and combinations of those -this. Examples of protein labels useful in the present invention include, but are not limited to, Glutathione-S-Transferase (GST) tags, Staphlococcus aureus protein A, Nus A, chitin binding protein (CBP), thioredoxin, maltose binding protein (MBP), carboxylated biotin transporter proteins (BCCP), constant fragment of immunoglobulins (Fc), labels comprising a fluorescent protein such as GFP (Green Fluorescent Protein), RFP (Red Fluorescent Protein), CFP (Cyan Fluorescent Protein) or YFP (Yellow Fluorescent Protein), and combinations thereof. According to a preferred embodiment, the fusion protein comprises a tag consisting of six histidines and / or one or more Flag patterns, preferably three Flag patterns. According to a particular embodiment, the fusion protein comprises a tag consisting of six histidines and three Flag motifs. The fusion protein as described above can be introduced into the cell in a protein form, especially in its mature form or in the form of a precursor, preferably in its mature form, or in the form of an acid. nucleic acid encoding said protein. When the fusion protein is introduced into the cell in a protein form, protecting groups may be added at the C- and / or N-termini to enhance the resistance of the fusion protein to peptidases. For example, the protecting group at the N-terminus may be acylation or acetylation and the protective group at the C-terminus may be amidation or esterification. The action of proteases can also be counteracted by the use of amino acids of configuration D, the cyclization of the protein by formation of disulfide bridges, lactam rings or bonds between the N- and C-termini. The fusion protein of the invention may also comprise pseudo-peptide bonds replacing the "conventional" CONH peptide bonds and conferring increased resistance to peptidases, such as CHOH-CH2, NHCO, CH2-O, CH2CH2, CO-CH2, NN, CH = CH, CH2NH, and CH2-S. The fusion protein may also comprise one or more amino acids which are rare amino acids, in particular hydroxyproline, hydroxylysine, allohydroxylysine, 6-N-methylsilyl, N-methylglycine, N-methylglycine, N-ethylasparagine allo-isoleucine, N-methylisoleucine, N-methylvaline, pyroglutamine, aminobutyric acid; or synthetic amino acids including ornithine, norleucine, norvaline and cyclohexyl-alanine. The fusion protein according to the invention can be obtained by conventional chemical synthesis (in the solid phase or in liquid homogeneous phase) or by enzymatic synthesis (Kullmann W, Enzymatic peptide synthesis, 1987, CRC Press, Florida). It can also be obtained by a method of culturing a host cell expressing a nucleic acid encoding the fusion protein and recovering said protein from these cells or from the culture medium. As used herein, the term "guide RNA" or "gRNA" refers to an RNA molecule capable of interacting with the Cas9 domain of the fusion protein to guide it to a target chromosomal region. Each gRNA comprises two regions: a first region (commonly referred to as "SDS" region), at the 5 'end of the gRNA, which is complementary to the target chromosomal region and mimics the endogenous CRISPR system CRRR, and a second region (commonly referred to as a "handle" region) at the 3 'end of the gRNA, which mimics the base-pairing interactions between the trans-activating crRNA and CRISPR endogenous and has a double-strand structure stem and loop terminating 3 'by a sequence essentially single-stranded. This second region is essential for binding the gRNA to the Cas9 domain of the fusion protein. The first region of the gRNA varies according to the targeted chromosomal sequence. On the other hand, the "handle" regions of the different gRNAs used may be identical or different.

The "SDS" region of the gRNA that is complementary to the target chromosomal region generally comprises between 10 and 25 nucleotides. Preferably, this region has a length of 19, 20 or 21 nucleotides, and particularly preferably 20 nucleotides. The second region of the gRNA has a stem and loop structure (or hairpin structure). The lengths of the stem and buckle may vary. Preferably, the loop has a length of 3 to 10 nucleotides and the stem a length of 6 to 20 nucleotides. The stem may optionally have unpaired regions (forming "bumps") of 1 to 10 nucleotides. Preferably, the overall length of this "handle" region is 50 to 100 nucleotides, and more preferably 82 nucleotides. The total length of a gRNA is generally from 50 to 140 nucleotides, preferably from 80 to 125 nucleotides, and more preferably from 90 to 110 nucleotides. According to a particular embodiment, a gRNA as used in the present invention is 102 nucleotides in length. The gRNA is preferably formed of a single RNA molecule comprising both domains. Alternatively, the gRNA can be formed of two distinct RNA molecules, the first molecule comprising the "SDS" region and half the stem of the second region, and the second molecule comprising the second half of the the gRNAs. Thus, the pairing of the two RNA molecules by their complementary sequences at the stem, forms a functional gRNA. Those skilled in the art can readily define the sequence and structure of the gRNAs according to the chromosomal region to be targeted using well known techniques (see for example the article by Di Carlo et al, Nucleic Acids Research 2013, 1-8). In the process according to the invention, one or more gRNAs can be used simultaneously. These different gRNAs can target identical or different chromosomal regions, preferably different. The gRNAs can be introduced into the eukaryotic cell in the form of mature gRNA molecules, in the form of precursors or in the form of one or more nucleic acids encoding said gRNAs. When the gRNA (s) are introduced into the cell directly in the form of RNA molecules (mature or precursors), these gRNAs may contain modified nucleotides or chemical modifications allowing them, for example, to increase their resistance to nucleases and thus to increase their life in the cell. They may in particular comprise at least one modified or non-natural nucleotide such as, for example, a nucleotide comprising a modified base, such as inosine, methyl-5-deoxycytidine, dimethylamino-5-deoxyuridine, deoxyuridine or diamino. -2,6-purine, bromo-5-deoxyuridine or any other modified base for hybridization. The gRNAs used according to the invention can also be modified at the level of the internucleotide linkage, for example phosphorothioates, H-phosphonates or alkylphosphonates, or at the level of the backbone such as, for example, alpha-oligonucleotides, 0-alkyl ribose or PNAs (Peptid Nucleic Acid) (Egholm et al., 1992 J. Am. Chem. Soc., 114, 1895-1897). The gRNAs can be natural, synthetic RNA or produced by recombinant techniques. These gRNAs can be prepared by any method known to those skilled in the art such as, for example, chemical synthesis, in vivo transcription or amplification techniques. According to one embodiment, the method comprises introducing into the eukaryotic cell the fusion protein and one or more gRNAs capable of targeting the action of the fusion protein to a given chromosomal region. The protein and the gRNAs can be introduced into the cytoplasm or nucleus of the eukaryotic cell by any method known to those skilled in the art, for example by microinjection. The fusion protein may in particular be introduced into the cell as part of a protein-RNA complex comprising at least one gRNA. In another embodiment, the method comprises introducing into the eukaryotic cell the fusion protein and one or more nucleic acids encoding one or more gRNAs. In yet another embodiment, the method comprises introducing into the eukaryotic cell a nucleic acid encoding the fusion protein and one or more gRNAs. In yet another embodiment, the method comprises introducing into the eukaryotic cell a nucleic acid encoding the fusion protein and one or more nucleic acids encoding one or more gRNAs. The fusion protein, or the nucleic acid encoding it, and the gRNA (s), or the nucleic acid (s) encoding it, may be introduced simultaneously or sequentially into the cell. In embodiments where the fusion protein and / or the gRNA (s) are introduced into the eukaryotic cell in the form of a nucleic acid encoding said protein and / or gRNA, the expression of said nucleic acids allows to produce the fusion protein and / or the gRNA (s) in the cell.

For the purposes of the invention, the term "nucleic acid" means any molecule based on DNA or RNA. It may be synthetic or semi-synthetic molecules, recombinant, possibly amplified or cloned in vectors, chemically modified, comprising non-natural bases or modified nucleotides comprising for example a modified bond, a modified purine or pyrimidine base, or a modified sugar. Preferably, the use of the codons is optimized according to the nature of the eukaryotic cell. The nucleic acids coding for the fusion protein and those encoding the gRNAs may be placed under the control of identical or different promoters, constitutive or inducible, in particular promoters specific for meiosis. According to a preferred embodiment, the nucleic acids are placed under the control of constitutive promoters such as the promoter ADH1 or the promoters pRPR1 and 5NR52 depending on the RNA polymerase III, more preferably the pRPR1 promoter. The nucleic acids encoding the fusion protein and the gRNA (s) may be arranged on the same construct, in particular on the same expression vector, or on separate constructs. Alternatively, the nucleic acids can be inserted into the genome of the eukaryotic cell into identical or distinct regions. According to a preferred embodiment, the nucleic acids encoding the fusion protein and the gRNA (s) are arranged on the same expression vector. The nucleic acids as described above can be introduced into the eukaryotic cell by any method known to those skilled in the art, in particular by microinjection, transfection, electroporation and biolistics. Optionally, expression or activity of the endogenous Spol 1 protein of the eukaryotic cell can be suppressed to better control meiotic recombination phenomena. This inactivation can be carried out by techniques well known to those skilled in the art, in particular by inactivating the gene encoding the endogenous Spoll protein or by inhibiting its expression by means of interfering RNA. After the introduction into the eukaryotic cell of the fusion protein and one or more gRNAs, or the nucleic acids encoding them, the method according to the invention comprises the induction of the entry into prophase I of meiosis. of said cell. This induction can be done according to different methods, well known to those skilled in the art. By way of example, when the eukaryotic cell is a mouse cell, the entry of meiotic prophase I cells may be induced by the addition of retinoic acid (Bowles J et al, 2006, Sciences, 312 (5773 ), pp. 596-600).

When the eukaryotic cell is a yeast, this induction can be carried out by the transfer of yeast from a rich medium to a sporulation medium, preferably free of fermentable carbon source or nitrogen, and the incubation of yeasts in the sporulation medium for a time sufficient to induce Spo 1 1-dependent double-strand breaks. The initiation of the meiotic cycle depends on several signals: the presence of the two sexual alleles MATa and MATa, the absence of a source of nitrogen and fermentable carbon. As used herein, the term "rich medium" refers to a culture medium comprising a fermentable carbon source and a nitrogen source as well as all the nutrients necessary for yeasts to multiply by mitotic division. This medium can be easily chosen by those skilled in the art and can, for example, be selected from the group consisting of YPD medium (1% yeast extract, 2% bactopeptone and 2% glucose), YPG medium (1% extract yeast, 2% bactopeptone and 3% glycerol) and a complete synthetic medium (or SC medium) (Treco and Lundblad, 2001, Mol Mol Biol., Chapter 13, Unit 13.1). As used herein, the term "sporulation medium" refers to any medium inducing meiosis prophase entry of yeast cells without vegetative growth, particularly to a culture medium lacking a carbon source. fermentable nitrogen source but including a source of metabolizable carbon by respiration such as acetate. This medium may be readily selected by those skilled in the art and may, for example, be selected from the group consisting of 1% KAc medium (Wu and Lichten, 1994, Science, 263, pp. 515-518), SPM medium. (Kassir and Simchen, 1991, Meth Enzymol., 194, 94-110) and sporulation media described in the Sherman article (Sherman, Meth Enzymol., 1991, 194, 3-21). According to a preferred embodiment, before being incubated in the sporulation medium, the cells are cultured during a few division cycles in a pre-sporulation medium so as to obtain efficient and synchronous sporulation. The pre-sporulation medium may be readily selected by those skilled in the art. This medium may be, for example, SPS medium (Wu and Lichten, 1994, Science, 263, pp. 515-518). The choice of media (rich medium, pre-sporulation medium, sporulation medium) depends on the physiological and genetic characteristics of the yeast strain, especially if this strain is auxotrophic for one or more compounds. Once the cell is engaged in prophase I of meiosis, the meiotic process can continue until it produces four daughter cells with the desired recombinations.

Alternatively, when the eukaryotic cell is a yeast, and in particular a yeast of the genus Saccharomyces, the cells can be replaced under growing conditions to resume a mitotic process. This phenomenon, called "return-to-growth" or "RTG" has been previously described in patent application WO 2014/083142 and occurs when the cells entered into meiosis in response to a nutritional deficiency are put back in the presence of a source. of carbon and nitrogen after the formation of dependent Spoll double-strand breaks but before the first meiosis division (Honigberg and Esposito, Proc Nat Sci USA, 1994, 91, 6559-6563). Under these conditions, they interrupt their progression in meiotic differentiation stages to resume a mitotic growth mode while inducing the recombinations sought during the repair of double-strand breaks caused by Spoll (Sherman and Roman, Genetics, 1963, 48, 255- 261, Esposito and Esposito, Proc Nat Acad Sci, 1974, 71, pp. 3172-3176, Zenvirth et al, Genes to Cells, 1997, 2, pp. 487498). The method may furthermore comprise obtaining the cell (s) exhibiting the desired recombination (s). The method according to the invention can be used in all applications where it is desirable to improve and control meiotic recombination phenomena. In particular, the invention makes it possible to associate, preferably, genetic traits of interest. This preferential association allows, on the one hand, to reduce the time necessary for their selection, on the other hand, to generate possible but unlikely natural combinations. Finally, according to the embodiment chosen, the organisms obtained by this process can be considered as non-genetically modified (non-GMO) organisms. According to another aspect, the present invention relates to a method for generating variants of a eukaryotic organism, with the exception of humans, preferably a yeast, in particular a yeast strain of industrial interest, comprising - the introduction in a cell of said organism: a) a fusion protein comprising a Cas9 domain and a Spo domain 11, or a nucleic acid encoding said fusion protein; and b) one or more guide RNAs, or one or more nucleic acids encoding said guide RNAs, said guide RNAs comprising a Cas9 domain binding RNA structure and a sequence complementary to a targeted chromosomal region; and - inducing meiosis prophase I entry of said cell; obtaining cells with the desired recombination (s) at the targeted chromosomal region (s); and the genesis of a variant of the organism from said recombinant cell. In this method, the term "variant" must be understood broadly, it refers to an organism having at least one genotypic or phenotypic difference relative to the parent organisms. The recombinant cells can be obtained by allowing the meiosis to continue until spores are obtained, or, in the case of yeasts, by returning the cells to growth conditions after the induction of double-strand breaks in order to regain control. mitotic process. The present invention also relates to a method for identifying or locating genetic information encoding a characteristic of interest in a eukaryotic cell genome, preferably a yeast, comprising: - introducing into the eukaryotic cell: a) a fusion protein comprising a Cas9 domain and a Spo1 domain 1, or a nucleic acid encoding said fusion protein; and b) one or more guide RNAs, or one or more nucleic acids encoding said guide RNAs, said guide RNAs comprising a Cas9 domain binding RNA structure and a sequence complementary to a targeted chromosomal region; and - inducing meiosis prophase I entry of said cell; obtaining cells with the desired recombination (s) at the targeted chromosomal region (s); and - analyzing the genotypes and phenotypes of the recombinant cells to identify or locate the genetic information encoding the characteristic of interest. Preferably, the characteristic of interest is a quantitative trait of interest (QTL). In another aspect, the present invention relates to a fusion protein comprising a Cas9 domain and a Spoll domain as described above. The present invention also relates to a nucleic acid encoding said fusion protein according to the invention. The nucleic acid according to the invention may be in the form of DNA and / or RNA, single-stranded or double-stranded. According to a preferred embodiment, the nucleic acid is an isolated DNA molecule, synthesized by recombinant techniques well known to those skilled in the art. The nucleic acid according to the invention can be deduced from the sequence of the fusion protein according to the invention and the use of the codons can be adapted according to the host cell in which the nucleic acid is to be transcribed. The present invention further relates to an expression cassette comprising a nucleic acid according to the invention operably linked to the sequences necessary for its expression. In particular, the nucleic acid may be under the control of a promoter allowing its expression in a host cell. In general, an expression cassette comprises, or consists of, a promoter for initiating transcription, a nucleic acid according to the invention, and a transcription terminator. The term "expression cassette" refers to a nucleic acid construct comprising a coding region and a regulatory region, operably linked. The term "operably linked" indicates that the elements are combined so that the expression of the coding sequence is under the control of the transcriptional promoter. Typically, the promoter sequence is placed upstream of the gene of interest, at a distance thereof compatible with the control of its expression. Spacer sequences may be present between the regulatory elements and the gene, provided that they do not prevent the expression. The expression cassette may also comprise at least one enhancer activator sequence operably linked to the promoter. A wide variety of promoters useful for the expression of genes of interest in cells or host organisms are available to those skilled in the art. They include constitutive promoters as well as inducible promoters that are activated or repressed by exogenous physical or chemical stimuli. Preferably, the nucleic acid according to the invention is placed under the control of a constitutive promoter or a specific promoter of meiosis. Examples of meiosis-specific promoters that can be used in the context of the present invention include, but are not limited to, endogenous promoters of Spo 11, Spoll partner promoters for the formation of double-strand breaks, the Rec8 promoter (Murakami). & Nicolas, 2009, Mol Cell Biol, 29, 3500-16,), or the Spo13 promoter (Malkova et al, 1996, Genetics, 143, 741-754,). Other inducible promoters can also be used such as the estradiol promoter (Carlie & Amon, 2008 Cell, 133, 280-91,), the methionine promoter (Care et al, 1999, Molecular Microb 34, 792-798,), promoters induced by thermal shocks, metals, steroids, antibiotics and alcohol.

Constitutive promoters that may be used in the context of the present invention are, by way of non-limiting examples: the promoter of the immediate cytomegalovirus (CMV) immediate genes, the simian virus (SV40) promoter, the adenovirus major late promoter, the Rous sarcoma virus (RSV) promoter, the mouse mammary tumor virus (MMTV) promoter, the phosphoglycerate kinase (PGK) promoter, the ED1-alpha elongation factor promoter, the promoters of the mouse ubiquitin, actin promoters, tubulin promoters, immunoglobulin promoters, alcohol dehydrogenase 1 (ADH1) promoter, RNA polymerase III-dependent promoters such as U6, U3 promoters, H1, 7SL, pRPR1 ("Ribonuclease P RNA 1") or SNR52 ("small nuclear RNA 52"). The transcription terminator can be readily selected by those skilled in the art. Preferably, this terminator is RPR1t or the 3 'flanking sequence of the Saccharomyces cerevisiae SUP4 gene. The present invention further relates to an expression vector comprising a nucleic acid or an expression cassette according to the invention. This expression vector can be used to transform a host cell and allow the expression of the nucleic acid according to the invention in said cell. The vectors can be constructed by conventional molecular biology techniques well known to those skilled in the art. Advantageously, the expression vector comprises regulatory elements allowing the expression of the nucleic acid according to the invention. These elements may comprise, for example, transcription promoters, transcription activators, terminator sequences, initiation and termination codons. The methods for selecting these elements based on the host cell in which expression is desired are well known to those skilled in the art. In a particular embodiment, the expression vector comprises a nucleic acid encoding the fusion protein according to the invention, placed under the control of a constitutive promoter, preferably the ADH1 promoter (pADH1). It may also include a terminator sequence such as the terminator ADH1 (tADH1). The expression vector may comprise one or more origins of replication, bacterial or eukaryotic. The expression vector may in particular comprise a functional origin of bacterial replication in E. coli such as the Co1E1 origin of replication. Alternatively, the vector may comprise a eukaryotic origin of replication, preferably functional in S. cerevisiae.

The vector may further comprise elements allowing its selection in a bacterial or eukaryotic host cell, such as, for example, an antibiotic resistance gene or a selection gene ensuring the complementation of the respective gene deleted in the genome of the host cell. Such elements are well known to those skilled in the art and widely described in the literature. In a particular embodiment, the expression vector comprises one or more antibiotic resistance genes, preferably an ampicillin resistance gene and / or a kanamycin resistance gene. The expression vector may also comprise one or more sequences allowing the targeted insertion of the vector, the expression cassette or the nucleic acid into the genome of a host cell. Preferably, the insertion is carried out at the level of a gene whose inactivation allows the selection of the host cells having integrated the vector, the cassette or the nucleic acid, such as the TRP 1 locus. The vector may be circular or linear, single- or double-stranded. It is advantageously chosen from plasmids, phages, phagemids, viruses, cosmids and artificial chromosomes. Preferably, the vector is a plasmid. The present invention relates in particular to a vector, preferably a plasmid, comprising an origin of bacterial replication, preferably the Co1E1 origin, a nucleic acid as defined above under the control of a promoter, preferably a constitutive promoter such as the ADH1 promoter, a terminator, preferably the ADH1 terminator, one or more selection markers, preferably resistance markers such as the kanamycin or ampicillin resistance gene, and one or more sequences allowing targeted insertion of the vector, expression cassette or nucleic acid into the genome of the host cell, preferably at the TRP 1 locus of the genome of a yeast. In a particular embodiment, the nucleic acid according to the invention carried by the vector codes for a fusion protein comprising one or more tags, preferably comprising a tag consisting of six histidines and / or one or more Flag motifs, of preferably three Flag patterns. Preferably the label or labels are C-terminal. According to one particular embodiment, the expression vector is the plasmid P1 of nucleotide sequence SEQ ID NO: 1 or the plasmid P2, of nucleotide sequence SEQ ID NO: 2. The present invention also relates to the use of a nucleic acid , an expression cassette or an expression vector according to the invention to transform or transfect a cell. The host cell may be transiently / stably transfected / transfected and the nucleic acid, cassette or vector may be contained in the cell as an episome or integrated into the genome of the host cell. The present invention relates to a host cell comprising a nucleic acid, a cassette or an expression vector according to the invention. Preferably, the cell is a eukaryotic cell, in particular a yeast cell, plant cell, fungus cell or an animal cell. Particularly preferably, the host cell is a yeast cell. In a particular embodiment, the host cell is non-human and / or non-embryonic. The present invention also relates to the use of the fusion protein, the nucleic acid, the expression cassette or the expression vector according to the invention for (i) inducing targeted meiotic recombination in a eukaryotic cell, (ii) generating variants of a eukaryotic organism; and / or (iii) identifying or locating genetic information encoding a characteristic of interest in a eukaryotic cell genome. The present invention also relates to a kit comprising a fusion protein, a nucleic acid, an expression cassette or an expression vector according to the invention, or a host cell transformed or transfected with a nucleic acid, an expression cassette. or an expression vector according to the invention. It also relates to the use of said kit for implementing a method according to the invention, in particular for (i) inducing targeted meiotic recombinations in a eukaryotic cell, (ii) generating variants of a eukaryotic organism, and / or (iii) identifying or locating genetic information encoding a characteristic of interest in a eukaryotic cell genome. The following examples are presented for illustrative and non-limiting purposes. EXAMPLES 1. Design, synthesis and cloning of a nucleotide sequence encoding SpCas9 protein and its SpCas9 * form which is deficient for nuclease activity. The SpCas9 gene encoding the Cas9 protein is derived from the bacterial strain Streptococcus pyogenes. The catalytically inactive form of SpCas9 (SpCas9 *) differs from that of SpCas9 by two point mutations: aspartate 10 and histidine 840 have both been substituted for alanines (Asp10-> A1a1 ° and His840-> A1a840). .

Due to variations in the frequency of use of genetic codons between Streptococcus pyogenes and Saccharomyces cerevisiae, the SpCas9 and SpCas9 * gene sequences have been adapted to optimize their expression in yeast (yeast optim SpCas9 and yeast optim SpCas9 *). The amino acid sequences of the two proteins have not been modified. 2. Engineering yeast optim SpCas9 and yeast optim SpCas9 * sequences in order to fuse SpCas9 and SpCas9 * proteins with meiotic transesterase Spoll. Engineering steps of the Yeast Optim SpCas9 and Yeast Optim SpCas9 * sequences were used to fuse the SpCas9 and SpCas9 * proteins with a nuclear localization signal (NLS) associated with a N-terminus linker (linker 1) and with a second C-terminus linker (linker 2) (which will separate the SpCas9 and SpCas9 * proteins from the Spoll protein in the final construct). The nucleotide sequences thus obtained and coding for the NLS -linker 1-SpCas9-linker 2 and NLS -linker 1-SpCas9 * -linker 2 protein sequences were then cloned into an integrative plasmid, containing the complete form of the Saccharomyces Spoll protein. cerevisiae labeled with a sequence coding for the double motif 6xHis3xFlag at C-terminus and whose expression is controlled by the constitutive promoter pADH1. The resultant plasmid constructs, P1 and P2, thus contained the N-terminus fusion of NLS-linker 1-SpCas9-linker2 and NLS-linker 1-SpCas9 * -linker2 to Spoll protein. Accordingly, P1 and P2 respectively allowed the constitutive expression in yeast of the NLS -SpCas9-Spo1-6xHis-3xFlag (SEQ ID NO: 6) and NLSSpCas9 * -Spo11-6xHis-3xFlag (SEQ ID NO: 7) (Figure 1). 3. Engineering of unique and multiple "guide" RNA expression vectors. From a 2 micron plasmid (Farzadfard F et al, 2013, ACS Synth Biol., 2, pp. 604-613, DiCarlo JE et al., 2013, Nucleic Acids Res., 41 (7), pp. 4336-4343) containing the "handle" region (82 nucleotides) of a "guide" RNA (gRNA), placed under the control of an RNA polymerase III-dependent constitutive promoter such as pRPR1 or SNR52, the Expression vector of a single 102 nucleotide gRNA was constructed by cloning the 20 nucleotide sequence determining gRNA specificity ("SDS" region) at a restriction site located immediately 5 'from the sequence encoding the linearized vector "handle" region, by the Gibson assembly method (Figure 2A) .This expression vector had a sequence with many unique sites of restriction (multiple cloning sites (MCS)) downstream of the terminator (RPR 1t or flanking sequence of 3 'of SUP4), so as to obtain a system allowing multiplexing targeting of In the meiotic recombination sites, several gRNA expression cassettes were introduced into the expression vector at its MCS. The gRNA expression cassettes are composed of a constitutive promoter dependent on the RNA polymerase III (pRPR1 or SNR52), the specific gRNA and a terminator (RPRlt or the flanking sequence of the 3 'of SUP4) . These gRNA expression cassettes were first cloned into single gRNA expression vectors (see above), then amplified by PCR before being cloned successively at the cloning site. multiple (MCS) expression vector of a single gRNA by conventional insertion / ligation techniques (Figure 2B). This strategy results in concatenating several gRNA cassettes into a single expression vector. 4. Co-expression of SpCas9-Spoll and SpCas9 * -Spo11 fusion proteins with yeast gRNAs. In order to introduce the NLS-SpCas9-Spol1 or NLS-SpCas9 * -Spo11 fusions into the TRP1 chromosomal locus, Saccharomyces cerevisiae yeast strains were transformed by thermal shock with the linearized Pl or P2 vectors. These fusion proteins carrying the 3xFlag C-terminal tag were placed under the control of the constitutive ADH1 promoter. After transformation, the cells were plated on petri dishes containing a selective medium (adapted to the selection markers carried by the plasmids P1 and P2) in order to select the transformants having integrated the fusion into their genome. The expression vector of the gRNA (s) was then introduced into diploid yeast strains expressing the NLS-SpCas9-Spol1 or NLS-SpCas9 * -Spo11 fusion proteins by heat shock transformation. The cells were then plated on selective medium for plasmid selection markers P1, P2 and 2 micron. The gRNA expression plasmids had a 2 micron origin of replication (20 which allowed them to maintain a high copy number in each yeast cell (50100 copies / cell).) Formation of meiotic double-strand breaks uniquely generated or multiplexed by the SpCas9-Spol1 or SpCas9 * -Spo11 fusion proteins at genomic sites targeted by single or multiple gRNAs is then detected by Southern blot analysis of genomic DNA extracted from diploid cells cultured in medium. sporulation.

Claims (9)

CLAIMS 1- A method for inducing targeted meiotic recombination in a eukaryotic cell comprising - introducing into said cell: a) a fusion protein comprising a Cas9 domain and a Spo111 domain or a nucleic acid encoding said protein. fusion; and b) one or more guide RNAs or one or more nucleic acids encoding said guide RNAs, said guide RNAs comprising a Cas9 domain binding RNA structure of the fusion protein and a sequence complementary to the targeted chromosomal region; and - inducing meiosis prophase 1 entry of said cell and wherein said eukaryotic cell is a yeast, plant or fungal cell. The method of claim 1, wherein the fusion protein further comprises a nuclear localization signal sequence. The method according to any one of the preceding claims, wherein the Cas9 domain is a Cas9 protein deficient for nuclease activity. A method according to any one of the preceding claims, wherein the expression of the nucleic acid encoding said fusion protein is under the control of a constitutive, inducible or meiosis specific promoter. The method according to any one of the preceding claims, further comprising introducing one or more additional guide RNAs targeting one or more other chromosomal regions, or nucleic acids encoding said additional guide RNAs. The method of any one of the preceding claims, wherein the cell is a yeast. The method according to claim 6, wherein the induction of prophase 1 entry is obtained by transfer of the yeast from a rich medium to a sporulation medium. Fusion protein as defined in any one of of the preceding claims. 9. Nucleic acid encoding the fusion protein according to claim 8. 10. Expression cassette or vector comprising a nucleic acid according to claim 9. The vector of claim 10, said vector being a plasmid comprising - a bacterial origin of replication, preferably the ColEl origin. an expression cassette comprising a nucleic acid as defined in claim 9 under the control of an expression promoter, preferably the ADH1 promoter, and a terminator, preferably the ADH1 'terminator; selection, preferably of resistance markers such as the kanamycin or ampicillin resistance gene, and / or one or more sequences allowing the targeted insertion of the vector, the expression cassette or the nucleic acid in the genome of a host cell, preferably at the TRPI locus of the genome of a yeast. The vector of claim 11, comprising, or consisting of, a nucleotide sequence selected from SEQ ID NO: 1 and SEQ ID NO: